Nanoparticles carrying antibiotics

ABSTRACT

The subject invention pertains to polyacrylate homopolymers produced from acrylolated drug monomers. The homopolymers can be produced in the form of nanoparticles. The nanoparticles comprising the homopolymers can be produced via a free radical-induced emulsion polymerization of the acrylolated drug monomers to produce an aqueous emulsion of uniformly sized nanoparticles. The homopolymers of the invention containing acrylolated antibiotic monomers can be active against Gram-positive and Gram-negative bacteria, such as Staphylococcus aureus and Escherichia coil. Accordingly, methods are provided of treating a disease, for example, an infection, by administering to a subject the homopolymers, homopolymeric nanoparticles, or emulsions containing homopolymeric nanoparticles of the invention.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.62/583,209, filed Nov. 8, 2017, the disclosure of which is herebyincorporated by reference in its entirety including any tables, figures,or drawings.

BACKGROUND OF THE INVENTION

The growing antibiotic resistance of harmful microbes, such asmethicillin-resistant Staphylococcus aureus (MRSA), has emerged as oneof the dominating concerns of today's public health system, causingscientists to look for ways to circumvent this resistance through drugdelivery methods and systems.

Aqueous polyacrylate nanoparticle emulsions for the purpose ofwater-solubilizing and encasing certain antibacterial compounds weredescribed as means to improve their stability and antibiotic activityespecially towards multi-drug resistant strains of bacteria. Thesenanoparticle emulsions were prepared through radical-induced emulsionpolymerization of butyl acrylate/styrene mixtures (7:3 w/w) in water at600° C., using sodium dodecyl sulfate (SDS) as an emulsifying agent andpotassium persulfate as a radical initiator. The reactions led to theformation of homogeneous, stable aqueous emulsions containinguniformly-sized nanoparticles of 45-50 nm in diameter. The method wassuccessfully applied to penicillins and N-thiolated β-lactams, such thatthe antibacterial agents could be introduced into the nanoparticleeither by non-covalent entrapment as a free drug, or covalently via anacryloyl derivative. The antibiotic-containing nanoparticles showedpromising in vitro activity against pathogenic bacteria such asmethicillin-resistant S. aureus (MRSA).

While these earlier nanoparticle emulsions provided increased watersolubility and, in some cases, improved bioactivity of the β-lactamantibacterial agent, the polyacrylate backbone was largely comprised ofnon-bioactive monomers (butyl acrylate-styrene or methylmethacrylate-styrene), and only 1-3% (by weight) of the antibacterialagent in the nanoparticle. The amount of drug loading into thenanoparticle during the assembly process was limited by how muchsurfactant could be used, given that amounts exceeding 3% (by weight) ofSDS caused discernable cytotoxicity. These emulsions contained up to 20%of solid content (as a mixture of nanoparticles and a small amount ofnon-emulsified polymers) and 0.2-1% of the antibacterial agent inside ofthe nanoparticles. The resulting emulsions were typically milky inconsistency and somewhat sticky when exposed to air, causing films torapidly form when dried. Also, unwanted coagulation within syringes,micro-porous filters, and gel columns made it very difficult to purifyand use them for in vivo testing. Purification techniques that enablethe removal of residual unreacted monomers and non-nanoparticleoligomers within the emulsion could address some of these issues.Therefore, polymeric nanoparticles that are soluble in water and containhigher amount of drugs are desirable.

BRIEF SUMMARY OF THE INVENTION

This disclosure describes polymers produced from acrylate monomers ofdrugs, particularly, polymers produced exclusively from acrylatemonomers of drugs, and surfactant combinations. The polymers disclosedherein contain higher amounts of drugs without increasing overallcytotoxicity or instability of the emulsion.

Certain embodiments of the invention provide methods of preparingnanoparticle emulsions containing an acrylolated drug as the solemonomer for producing polymeric nanoparticle emulsion. In preferredembodiments, the drug can be chemically modified to connect an acrylategroup to the drug, i.e., acrylolated. In even more preferredembodiments, the drug is an antibiotic, for example, ciprofloxacin.

Further embodiments of the invention provide methods of producingpolymeric nanoparticle emulsions containing an acrylolated drug as thesole monomer for producing the polymeric nanoparticle emulsion. Certainsuch methods comprise pre-solubilization of a water-insoluble drug, forexample, an antibacterial agent, in an organic solvent to permit moreuniform addition into the aqueous solution and to form homogeneousemulsions. In preferred embodiments, dichloromethane is used tosolubilize the monomeric acrylolated drug, e.g., monomeric acrylolatedciprofloxacin.

In additional embodiments, increased temperature of about 90° C. (ratherthan 75° C.) for polymerization, increased stir speed of about 1100 rpm(rather than 750 rpm), and the addition of a surfactant, such as sodiumdodecyl sulfate, before adding the monomers can be performed to furtherfacilitate evaporation of an organic solvent. In further embodiments,the polymerization reactions are run for about 48 hours (rather than theusual 6 hours).

Further embodiments of the invention provide methods of treating adisease in a subject, for example, an infection, by administering to thesubject the homopolymers of acrylolated drugs, nanoparticles comprisingthe homopolymers of acrylolated drugs, or emulsions containing thenanoparticles of the homopolymers of the acrylolated drugs of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Scheme for preparing poly(acrylate-styrene) emulsions.

FIG. 2. Co-monomer-based encapsulation of antibiotic into polyacrylatenanoparticle emulsions.

FIG. 3. Removal of co-monomers and formation of polymer via 100% of theacrylate antibiotic analog.

FIG. 4. Scheme for synthesis of N-acryloylciprofloxacin.

FIG. 5. Scheme for poly(N-acryloylciprofloxacin) nanoparticle emulsions.

FIG. 6. On the left, an example of a successful emulsion. On the right,two examples of unsuccessful emulsions.

FIG. 7. Size of emulsified nanoparticles vs the % concentration ofN-acryloyl ciprofloxacin in the emulsions.

FIG. 8. Scanning electron microscope image of the dried emulsion.

FIG. 9. A zoomed-in SEM image of a potential micelle within theemulsion.

DETAILED DISCLOSURE OF THE INVENTION

As used herein, the singular forms “a,” “an,” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. Furthermore, to the extent that the terms “including,”“includes,” “having,” “has,” “with,” or variants thereof are used ineither the detailed description and/or the claims, such terms areintended to be inclusive in a manner similar to the term “comprising.”The transitional terms/phrases (and any grammatical variations thereof)“comprising,” “comprises,” “comprise,” “consisting essentially of,”“consists essentially of,” “consisting,” and “consists” can be usedinterchangeably.

The phrases “consisting essentially of” or “consists essentially of”indicate that the claim encompasses embodiments containing the specifiedmaterials or steps and those that do not materially affect the basic andnovel characteristic(s) of the claim.

The term “about” means within an acceptable error range for theparticular value as determined by one of ordinary skill in the art,which will depend in part on how the value is measured or determined,i.e., the limitations of the measurement system. For example, where theterm “about” is used to describe compositions containing amounts ofingredients or a temperature or a rate of stirring, these parameters canbe varied between 0% and 10% around the stated value (X±10%).

In the present disclosure, ranges are stated in shorthand, so as toavoid having to set out at length and describe each and every valuewithin the range. Any appropriate value within the range can beselected, where appropriate, as the upper value, lower value, or theterminus of the range. For example, a range of 0.1-1.0 represents theterminal values of 0.1 and 1.0, as well as the intermediate values of0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and all intermediate rangesencompassed within 0.1-1.0, such as 0.2-0.5, 0.2-0.8, 0.7-1.0, etc.Values having at least two significant digits within a range areenvisioned, for example, a range of 5-10 indicates all the valuesbetween 5.0 and 10.0 as well as between 5.00 and 10.00 including theterminal values. When ranges are used herein combinations andsubcombinations of ranges (e.g., subranges within the disclosed range)and specific embodiments therein are intended to be explicitly included.

“Pharmaceutically acceptable carrier” or “pharmaceutically acceptableexcipient” includes any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents and the like. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theantigen in the vaccine, its use in the vaccine compositions of theinvention is contemplated.

“Treatment” (and grammatical variants of these terms), as used herein,are used interchangeably. These terms refer to an approach for obtainingbeneficial or desired results including but not limited to therapeuticbenefit. A therapeutic benefit is achieved with the eradication oramelioration of one or more of the physiological symptoms associatedwith the underlying disorder such that an improvement is observed in thepatient, notwithstanding that the patient may still be afflicted withthe underlying disorder.

The term “therapeutically effective amount” refers to that amount of adrug that is sufficient to effect the intended application including butnot limited to disease treatment. The therapeutically effective amountmay vary depending upon the intended application or the subject anddisease condition being treated, e.g., the weight and age of thesubject, the severity of the disease condition, the manner ofadministration and the like, which can readily be determined by one ofordinary skill in the art. The specific dose will vary depending on theparticular compounds chosen, the dosing regimen to be followed, whetherit is administered in combination with other compounds, timing ofadministration, the tissue to which it is administered, and the physicaldelivery system in which it is carried.

“Subject” refers to an animal, such as a mammal, for example a human.The methods described herein can be useful in both humans and non-humananimals. In some embodiments, the subject is a mammal (such as an animalmodel of disease), and in some embodiments, the subject is a human.Typical subjects include canine, feline, porcine, bovine, equine, andprimate.

A homopolymer refers to a polymer consisting of only one type ofmonomer. A co-polymer refers to a polymer containing more than one typeof monomers.

This disclosure provides polymers and nanoparticles containing suchpolymers. Certain polymers disclosed herein are produced bypolymerization of therapeutic monomers. The polymers and nanoparticlescontaining such polymers can be produced in the form of an emulsion witha surfactant, such as sodium dodecyl sulfate (SDS).

Accordingly, certain embodiments of the invention provide a homopolymerof an acrylolated drug as a monomer. Acrylolation of a drug can beperformed on a suitable atom in the drug, such as, C, S, O, and N,preferably, N.

In preferred embodiments, the drug is an antibiotic, such asciprofloxacin. The antibiotic compounds can belong to a class ofpenicillins, N-thiolated β-lactams, or fluoroquinolones. The β-lactamantibiotic can be a penicillin, penams, cephalosporin, monobactam, orcarbapenem. Non-limiting examples of β-lactam antibiotics includebenzylpenicillin, benzathine benzylpenicillin, procainebenzylpenicillin, phenoxymethylpenicillin (V), propicillin,pheneticillin, azidocillin, clometocillin, penamecillin, cloxacillin,dicloxacillin, flucloxacillin, oxacillin, nafcillin, methicillin,amoxicillin, ampicillin, pivampicillin, hetacillin, bacampicillin,metampicillin, talampicillin, epicillin, ticarcillin, carbenicillin,carindacillin, temocillin, piperacillin, azlocillin, mezlocillin,mecillinam, pivmecillinam, sulbenicillin, faropenem, ritipenem,ertapenem, antipseudomonal, doripenem, imipenem, meropenem, biapenem,panipenem, cefazolin, cefalexin, cefadroxil, cefapirin, cefazedone,cefazaflur, cefradine, cefroxadine, ceftezole, cefaloglycin,cefacetrile, cefalonium, cfaloridine, cefalotin, cefatrizine, cefaclor,cefotetan, cephamycin, cefoxitin, cefprozil, cefuroxime, cefuroximeaxetil, cefamandole, cefminox, cefonicid, ceforanide, cefotiam,cefbuperazone, cefuzonam, cefmetazole, carbacephem, loracarbef,cefixime, ceftriaxone, antipseudomonal, ceftazidime, cefoperazone,cefdinir, cefcapene, cefdaloxime, ceftizoxime, cefmenoxime, cefotaxime,cefpiramide, cefpodoxime, ceftibuten, cefditoren, cefetamet, cefodizime,cefpimizole, cefsulodin, cefteram, ceftiolene, oxacephem, flomoxef,latamoxef, cefepime, cefozopran, cefpirome, cefquinome, ceftarolinefosamil, ceftolozane, ceftobiprole, ceftiofur, cefquinome, cefovecin,aztreonam, tigemonam, carumonam, and nocardicin A.

Non-limiting examples of fluoroquinolone include enoxacin,ciprofloxacin, norfloxacin, ofloxacin, levofloxacin, trovafloxacin,gatifloxacin, or moxifloxacin.

A person of ordinary skill in the art can envision that any drug thatcan be acrylolated to produce the homopolymers according to thisdisclosure and such embodiments are within the purview of the invention.

In preferred embodiments, a homopolymer of an acrylolated drug is in theform of nanoparticles. In preferred embodiments, the nanoparticle have asize of between 600 nm and 1000 nm, preferably, between 700 nm and 1000nm, even more preferably, between 800 nm and 1000 nm, and mostpreferably, between 900 nm and 1000 nm. In a particular embodiment, thenanoparticles have a size of about 970 nm.

In other embodiments, the nanoparticles further comprise a detergent. Inmore preferred embodiments, the nanoparticles containing a detergent arein the form of an aqueous emulsion. Non-limiting examples of detergentsthat can be included in the nanoparticles or the emulsions disclosedherein include sodium dodecyl sulfate, cetyltrimethylammonium bromide,3-(N,N-dimethylmyristylammonio)propanosulfonate, dedecanoic acid2-(2-hydroxyethoxy)ethyl ester, sodium 11-(acrylolyloxyundecan-1-yl)sulfate,N-(11-Acryloyloxyundecyl)-N-(2-hydroxyethyl)-N,N-dimethylammoniuimbromide, N-(11-Acryloyloxyundecyl)-N,N-dimethyl-N-ethylammonium bromide,3-[N,N-Diethyl-N-(3-sulfopropyl)ammonio] acrylate,2(2-Acryloyloxyethoxy)ethyl dodecanoate, or any mixture thereof.

Certain other embodiments of the invention provide a co-polymer of twoor more acrylolated drugs. Such co-polymers can be useful foradministering a combination of drugs in one composition. A person ofordinary skill in the art can select appropriate combination of two ormore drugs that can be acrylolated and formed into a co-polymer. Muchlike the homopolymers described herein, such co-polymers can also be inthe form of nanoparticles comprising the co-polymers of acrylolateddrugs, or emulsions containing the nanoparticles of the co-polymers ofacrylolated drugs of the invention. Further, the methods describedherein for producing homopolymers of acrylolated drugs can be readilymodified to produce the co-polymers of two or more acrylolated drugs bysimply mixing appropriate amounts of monomers of the correspondingacrylolated drugs.

Specific embodiments of the invention provide a method of producing anaqueous emulsion of a homopolymer of an acrylate monomer of a drug. Suchmethod comprises the steps of:

a) dissolving the acrylolated monomer of the drug in an organic solvent;

b) dissolving a detergent in water;

c) mixing the solution of detergent in water with the solution of theacrylolated monomer of the drug in the organic solvent;

d) increasing the temperature of the mixture produced in step c);

e) contacting the mixture produced in step d) to an oxidant thatinitiates polymerization of the acrylate monomers of the drug; andoptionally, additional water;

f) stirring the mixture produced in step e) to produce the aqueousemulsion of the homopolymer of the acrylate monomer of the drug.

In certain embodiments, the step of dissolving a detergent in water isperformed at a temperature between 25° C. and 45° C., preferably, at atemperature between 30° C. and 40° C., more preferably, at a temperatureof about 35° C.

In preferred embodiments, the step of increasing the temperature of themixture produced in step c) is performed under constant stirring. Thetemperature of the mixture produced in step c) can be increased tobetween 80° C. and 110° C., preferably, between 85° C. and 105° C., evenmore preferably, between 90° C. and 100° C., and most preferably, toabout 95° C.

A person of ordinary skill in the art can select a suitable organicsolvent to dissolve an acrylolated drug. Organic solvents that can beused include methanol, ethanol, propylene glycol, hexane, glycerol,ethyl acetate, dichloromethane, or any mixture thereof. An organicsolvent is selected that would evaporate at a temperature of between 80°C. and 110° C. and thus, can be removed from the mixture during theprocess of making the aqueous emulsion. Additional examples of organicsolvents that can be used in the methods of the invention are well knownto a person of ordinary skill in the art and such embodiments are withinthe purview of the invention.

A detergent suitable for use in the methods of the invention includesodium dodecyl sulfate, cetyltrimethylammonium bromide,3-(N,N-dimethylmyristylammonio)propanosulfonate, dedecanoic acid2-(2-hydroxyethoxy)ethyl ester, sodium 11-(acrylolyloxyundecan-1-yl)sulfate,N-(11-Acryloyloxyundecyl)-N-(2-hydroxyethyl)-N,N-dimethylammoniuimbromide, N-(11-Acryloyloxyundecyl)-N,N-dimethyl-N-ethylammonium bromide,3-[N,N-Diethyl-N-(3-sulfopropyl)ammonio]acrylate,2(2-Acryloyloxyethoxy)ethyl dodecanoate, or any mixture thereof.Additional examples of detergents that can be used in the methods of theinvention are well known to a person of ordinary skill in the art andsuch embodiments are within the purview of the invention.

In preferred embodiments, dissolving an acrylate monomer of a drug in anorganic solvent is performed at a temperature of between 35° C. and 45°C., i.e., at a temperature higher than the room temperature. Suchtemperature facilitates dissolution of the drug in the organic solvent.

The step of increasing the temperature of the mixture produced in stepc) to between 80° C. and 100° C. is performed at a rate of between 5° C.and 15° C. per hour, preferably, at a rate of between 6° C. and 14° C.per hour, more preferably, at a rate of between 7° C. and 13° C. perhour, even more preferably, at a rate of between 8° C. and 12° C. perhour, and most preferably, at a rate of between 9° C. and 11° C. perhour, and particularly, at a rate of about 10° C. per hour. Such slowincrease in temperature ensures proper formation of homopolymers whileslowly evaporating the organic solvent out of the mixture, thusproducing an aqueous suspension of the homopolymeric nanoparticles.

Evaporation of the organic solvent from the mixture produced in step d)can be further facilitated by constant stirring, for example, at a rateof between 800 rpm to 1300 rpm, preferably, at a rate of between 900 rpmto 1200 rpm, more preferably, at a rate of between 1000 to 1100 rpm, andeven more preferably, at a rate of 1100 rpm.

Preferably, the oxidant that initiates polymerization is a radicalinitiator. Examples of oxidant that initiates polymerization of theacrylate monomers include potassium persulfate.

In preferred embodiments, the step of polymerization and stirring (stepse) and f) above) are carried out for between 36 to 60 hours, preferably,between 40 to 56 hours, more preferably, between 44 to 52 hours, andmost preferably, for about 48 hours.

In certain embodiments, the drug used in the methods of the invention isan antibiotic. Examples of antibiotics mentioned above in connectionwith the homopolymers of the invention can also be used in the methodsof the invention. Also, additional drugs or antibiotics that can be usedin the methods of the invention can be readily identified by a person ofordinary skill in the art and such embodiments are within the purview ofthe invention. Further, for co-polymers containing a combination ofdrugs or a combination of antibiotics, a person of ordinary skill in theart can select appropriate combinations of drugs based on intendedapplications.

The homopolymers, nanoparticles containing such homopolymers, andemulsions containing the nanoparticles disclosed herein exhibit theactivity of the drug, for example, the antibiotic used to produce thehomopolymer. Accordingly, certain embodiments of the invention provide amethod of treating a disease in a subject by administering atherapeutically effective amount of a homopolymers, nanoparticlescontaining such homopolymers, or emulsions containing the nanoparticlesdisclosed herein.

Co-polymers of a combination of acrylolated drugs, nanoparticlescontaining such co-polymers, and emulsions containing the nanoparticlesof such co-polymers can also be in the methods of treating a diseasedisclosed herein.

In preferred embodiments, the disease is an infection caused by aninfectious agent and the homopolymer is produced from an acrylolatedantibiotic. Accordingly, certain embodiments of the invention providemethods of treating an infection in a subject caused by an infectiousagent, the method comprising administering to the subject thehomopolymers, nanoparticles containing such homopolymers, or emulsionscontaining the nanoparticles disclosed herein. The homopolymers,nanoparticles, or emulsions disclosed herein can be administered in theform of a pharmaceutical composition comprising pharmaceuticallyacceptable carriers.

The polymers (including homopolymers and co-polymers disclosed herein),nanoparticles, or emulsions can be administered via, for example, oral,pulmonary, buccal, suppository, intravenous, intraperitoneal,intranasal, intramuscular or subcutaneous routes. Additional routes ofadministration are well known to a skilled artisan and such embodimentsare within the purview of this invention. The appropriate route ofadministration depends on the type of disease being treated, the subjectbeing treated, the stage and severity of the disease, etc. A person ofordinary skill in the art can determine an appropriate route ofadministration based on specific parameters.

In certain embodiments, the disease is an infection caused by a virus,bacterium, protozoan, helminth, archaebacterial, or a fungus. A personof ordinary skill in the art can select an appropriate drug orcombination of drugs to treat an infection and produce the correspondingpolymers, nanoparticles, or emulsions based on this disclosure.

Routes of Administration and Dosage Forms

In certain embodiments, the polymers, nanoparticles, or emulsions can beadministered intramuscularly, subcutaneously, intrathecally,intravenously or intraperitoneally by infusion or injection. Solutionsof the polymers, nanoparticles, or emulsions can be prepared in water,optionally mixed with a nontoxic surfactant. Under ordinary conditionsof storage and use, these preparations can contain a preservative toprevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions comprising thepolymers, nanoparticles, or emulsions, that are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions. Preferably, the ultimate dosage form should be sterile,fluid, and stable under the conditions of manufacture and storage. Theliquid carrier or vehicle can be a solvent or liquid dispersion mediumcomprising, for example, water, ethanol, a polyol (for example,glycerol, propylene glycol, liquid polyethylene glycols, and the like),vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof.The proper fluidity can be maintained by, for example, the formation ofliposomes, by the maintenance of the required particle size in the caseof dispersions, or by the use of surfactants. The prevention of theaction of microorganisms can be brought about by various antibacterialand antifungal agents, for example, parabens, chlorobutanol, phenol,sorbic acid, thimerosal, and the like. In many cases, it will bepreferable to include isotonic agents, for example, sugars, buffers, orsodium chloride. Prolonged absorption of the injectable compositions canbe brought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the polymers,nanoparticles, or emulsions in the required amount in the appropriatesolvent as described herein with various of the other ingredientsenumerated herein, as required, preferably followed by filtersterilization. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze drying techniques, which yield a powder thepolymers, nanoparticles, or emulsions plus any additional desiredingredient present in the previously sterile-filtered solutions.

The compositions of the subject invention may also be administeredorally, in combination with a pharmaceutically acceptable vehicle suchas an inert diluent or an assimilable edible carrier. They may beenclosed in hard or soft shell gelatin capsules, may be compressed intotablets, or may be incorporated directly with the food of the subject'sdiet.

For oral therapeutic administration the polymers, nanoparticles, oremulsions can be combined with one or more excipients and used in theform of ingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. The percentage of thepolymers, nanoparticles, or emulsions present in such compositions andpreparations can be varied can be conveniently be between about 2% toabout 60% of the weight of a given unit dosage form. The amount of thepolymers, nanoparticles, or emulsions in such therapeutically usefulcompositions is such that an effective dosage level will be obtained.

The tablets, troches, pills, capsules, and the like may also contain oneor more of the following: binders such as gum tragacanth, acacia, cornstarch or gelatin; excipients such as dicalcium phosphate; adisintegrating agent such as corn starch, potato starch, alginic acid,and the like; a lubricant such as magnesium stearate; and a sweeteningagent such as sucrose, fructose, lactose, or aspartame, or a flavoringagent such as peppermint, oil of wintergreen, or cherry flavoring may beadded.

When the unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol.

Various other materials may be present as coatings or for otherwisemodifying the physical form of the solid unit dosage form. For instance,tablets, pills, or capsules may be coated with gelatin, wax, shellac, orsugar, and the like. A syrup or elixir can contain the polymers,nanoparticles, or emulsions and sucrose or fructose as a sweeteningagent, methyl and propylparabens as preservatives, a dye, and flavoringsuch as cherry or orange flavor.

Any material used in preparing any unit dosage form should bepharmaceutically acceptable and substantially non-toxic in the amountsemployed.

In addition the polymers, nanoparticles, or emulsions can beincorporated into sustained-release preparations and devices. Forexample, the polymers, nanoparticles, or emulsions can be incorporatedinto time release capsules, time release tablets, time release pills,and time release polymers or nanoparticles.

Pharmaceutical compositions for topical administration of the polymers,nanoparticles, or emulsions to the epidermis (mucosal or cutaneoussurfaces) can be formulated as ointments, creams, lotions, gels, or as atransdermal patch. Such transdermal patches can contain penetrationenhancers such as linalool, carvacrol, thymol, citral, menthol,t-anethole, and the like. Ointments and creams can, for example, includean aqueous or oily base with the addition of suitable thickening agents,gelling agents, colorants, and the like. Lotions and creams can includean aqueous or oily base and typically also contain one or moreemulsifying agents, stabilizing agents, dispersing agents, suspendingagents, thickening agents, coloring agents, and the like. Gelspreferably include an aqueous carrier base and include a gelling agentsuch as cross-linked polyacrylic acid polymer, a derivatizedpolysaccharide (e.g., carboxymethyl cellulose), and the like.

Pharmaceutical compositions suitable for topical administration in themouth (e.g., buccal or sublingual administration) include lozengescomprising the composition in a flavored base, such as sucrose, acacia,or tragacanth; pastilles comprising the composition in an inert basesuch as gelatin and glycerin or sucrose and acacia; and mouthwashescomprising the active ingredient in a suitable liquid carrier. Thepharmaceutical compositions for topical administration in the mouth caninclude penetration enhancing agents, if desired.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina, and the like. Other solidcarriers include nontoxic polymeric nanoparticles or microparticles.Useful liquid carriers include water, alcohols, or glycols, orwater/alcohol/glycol blends, in which the polymers, nanoparticles, oremulsions can be dissolved or dispersed at effective levels, optionallywith the aid of non-toxic surfactants. Adjuvants such as fragrances andadditional antimicrobial agents can be added to optimize the propertiesfor a given use. The resultant liquid compositions can be applied fromabsorbent pads, used to impregnate bandages and other dressings, orsprayed onto the affected area using pump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses, or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user.

Examples of useful dermatological compositions that can be used todeliver the polymers, nanoparticles, or emulsions to the skin are knownin the art; for example, see Jacquet et al. (U.S. Pat. No. 4,608,392),Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157)and Wortzman (U.S. Pat. No. 4,820,508), all of which are herebyincorporated by reference.

The concentration of the polymers, nanoparticles, or emulsions in suchformulations can vary widely depending on the nature of the formulationand intended route of administration. For example, the concentration ofthe homopolymers, nanoparticles, or emulsions in a liquid composition,such as a lotion, can preferably be from about 0.1-25% by weight, or,more preferably, from about 0.5-10% by weight. The concentration in asemi-solid or solid composition such as a gel or a powder can preferablybe about 0.1-5% by weight, or, more preferably, about 0.5-2.5% byweight.

Pharmaceutical compositions for spinal administration or injection intoamniotic fluid can be provided in unit dose form in ampoules, pre-filledsyringes, small volume infusion, or in multi-dose containers, and caninclude an added preservative. The compositions for parenteraladministration can be suspensions, solutions, or emulsions, and cancontain excipients such as suspending agents, stabilizing agents, anddispersing agents.

A pharmaceutical composition suitable for rectal administrationcomprises the polymers, nanoparticles, or emulsions in combination witha solid or semisolid (e.g., cream or paste) carrier or vehicle. Forexample, such rectal compositions can be provided as unit dosesuppositories. Suitable carriers or vehicles include cocoa butter andother materials commonly used in the art.

According to one embodiment, pharmaceutical compositions of the presentinvention suitable for vaginal administration are provided as pessaries,tampons, creams, gels, pastes, foams, or sprays containing the polymers,nanoparticles, or emulsions in further combination with carriers knownin the art. Alternatively, compositions suitable for vaginaladministration can be delivered in a liquid or solid dosage form.

Pharmaceutical compositions suitable for intra-nasal administration arealso encompassed by the present invention. Such intra-nasal compositionscomprise the polymers, nanoparticles, or emulsions in a vehicle andsuitable administration device to deliver a liquid spray, dispersiblepowder, or drops. Drops may be formulated with an aqueous or non-aqueousbase also comprising one or more dispersing agents, solubilizing agents,or suspending agents. Liquid sprays are conveniently delivered from apressurized pack, an insufflator, a nebulizer, or other convenient meansof delivering an aerosol comprising the polymers, nanoparticles, oremulsions. Pressurized packs comprise a suitable propellant such asdichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide, or other suitable gas as iswell known in the art. Aerosol dosages can be controlled by providing avalve to deliver a metered amount of the homopolymers, nanoparticles, oremulsions.

The polymers, nanoparticles, or emulsions can be combined with an inertpowdered carrier and inhaled by the subject or insufflated.

Pharmaceutical compositions for administration by inhalation orinsufflation can be provided in the form of a dry powder composition,for example, a powder mix of the homopolymers, nanoparticles, oremulsions and a suitable powder base such as lactose or starch. Suchpowder composition can be provided in unit dosage form, for example, incapsules, cartridges, gelatin packs, or blister packs, from which thepowder can be administered with the aid of an inhalator or insufflator.

The exact amount (effective dose) of the polymers, nanoparticles, oremulsions administered can vary from subject to subject, depending on,for example, the species, age, weight, and general or clinical conditionof the subject, the severity or mechanism of any disorder being treated,the particular agent or vehicle used, the method and scheduling ofadministration, and the like. A therapeutically effective dose can bedetermined empirically, by conventional procedures known to those ofskill in the art. See, e.g., The Pharmacological Basis of Therapeutics,Goodman and Gilman, eds., Macmillan Publishing Co., New York. Forexample, an effective dose can be estimated initially either in cellculture assays or in suitable animal models. The animal model may alsobe used to determine the appropriate concentration ranges and routes ofadministration. Such information can then be used to determine usefuldoses and routes for administration in humans. Methods for theextrapolation of effective dosages in mice and other animals to humansare known to the art; for example, see U.S. Pat. No. 4,938,949, which ishereby incorporated by reference. A therapeutic dose can also beselected by analogy to dosages for comparable therapeutic agents.

The particular mode of administration and the dosage regimen will beselected by the attending clinician, taking into account the particularsof the case (e.g., the subject, the disease, the disease state involved,and whether the treatment is prophylactic). Treatment may involve dailyor multi-daily doses of compound(s) over a period of a few days tomonths, or even years.

In general, however, a suitable dose will be in the range of from about0.001 to about 100 mg/kg of body weight per day, preferably from about0.01 to about 100 mg/kg of body weight per day, more preferably, fromabout 0.1 to about 50 mg/kg of body weight per day, or even morepreferred, in a range of from about 1 to about 10 mg/kg of body weightper day. For example, a suitable dose may be about 1 mg/kg, 10 mg/kg, or50 mg/kg of body weight per day.

The polymers, nanoparticles, or emulsions can be convenientlyadministered in unit dosage form, containing for example, about 0.05 toabout 10000 mg, about 0.5 to about 10000 mg, about 5 to about 1000 mg,or about 50 to about 500 mg of the homopolymers, nanoparticles, oremulsions.

The polymers, nanoparticles, or emulsions can be administered to achievepeak plasma concentrations of, for example, from about 0.25 to about 200μM, about 0.5 to about 75 μM, about 1 to about 50 μM, about 2 to about30 μM, or about 5 to about 25 μM of each of the drug. Exemplarydesirable plasma concentrations include at least 0.25, 0.5, 1, 5, 10,25, 50, 75, 100 or 200 μM. For example, plasma levels may be from about1 to about 100 micromolar or from about 10 to about 25 micromolar. Thismay be achieved, for example, by the intravenous injection of a 0.05 to5% solution of the polymers, nanoparticles, or emulsions in saline, ororally administered as a bolus containing about 1 to about 100 mg of thehomopolymers, nanoparticles, or emulsions. Desirable blood levels may bemaintained by continuous or intermittent infusion.

The polymers, nanoparticles, or emulsions can be included in thecompositions within a therapeutically useful and effective concentrationrange, as determined by routine methods that are well known in themedical and pharmaceutical arts. For example, a typical composition caninclude the polymers, nanoparticles, or emulsions at a concentration inthe range of at least about 1 mg/ml, preferably at least about 4 mg/ml,more preferably at least 5 mg/ml and most preferably at least 6 mg/ml.

The polymers, nanoparticles, or emulsions can conveniently be presentedin a single dose or as divided doses administered at appropriateintervals, for example, as one dose per day or as two, three, four ormore sub-doses per day. The sub-dose itself may be further divided,e.g., into a number of discrete loosely spaced administrations; such asmultiple inhalations from an insufflator.

Optionally, the pharmaceutical compositions of the present invention caninclude one or more other therapeutic agents, e.g., as a combinationtherapy. The additional therapeutic agent(s) will be included in thecompositions within a therapeutically useful and effective concentrationrange, as determined by routine methods that are well known in themedical and pharmaceutical arts. The concentration of any particularadditional therapeutic agent may be in the same range as is typical foruse of that agent as a monotherapy, or the concentration may be lowerthan a typical monotherapy concentration if there is a synergy whencombined with the homopolymers, nanoparticles, or emulsions.

Following example illustrates procedures for practicing the invention.This example should not be construed as limiting.

Example 1 Polyacrylate Nanoparticle Emulsions: Forming Homo Poly(N-Acryloylciprofloxacin) as an Antibacterial Polymer Emulsion

The possibility of removing all non-bioactive monomers from nanoparticleconstruction was explored. Avoiding the use of co-monomers during theemulsion polymerization procedure allows for greater amount of loadingof the bioactive antibacterial monomer, producing a homopolymernanoparticle emulsion composed solely of the antibiotic monomer.

This Example delves into tackling the issue of limited loading ofbioactive compounds, and the need for a better carrier polymer to bindor encapsulate the drug for delivery. The surfactant has a limit of howmany organic/hydrophobic compounds it can contain within the micelleduring emulsion polymerization. As a result, the maximum amount oforganic content of the final emulsion is typically in the range of15-20% by weight. This restricts the usefulness of the nanoparticle asan effective drug carrier to 20% or less of the emulsion amount.

Polyacrylate nanoparticle emulsions can be easily prepared throughradical-induced emulsion polymerization of butyl acrylate/styrenemixtures (7:3 w/w) in water at 78° C., using sodium dodecyl sulfate(SDS) as an emulsifying agent and potassium persulfate as a radicalinitiator (FIG. 1). The reactions led to the formation of a homogeneous,stable aqueous emulsion containing uniformly-sized nanoparticles of45-50 nm in diameter. The method was successfully applied to penicillinsand N-thiolated β-lactams, in which the antibacterial agents could beintroduced into the nanoparticle either by non-covalent entrapment as afree drug, or covalently via their acryloyl derivative.

While these earlier nanoparticle emulsions provided increased watersolubility and, in some cases, improved bioactivity of the β-lactamantibacterial agent, the polyacrylate backbone was largely comprised ofthe non-bioactive monomers (butyl acrylate-styrene or methylmethacrylate-styrene (20% by weight of the emulsion), and thus only 1-3%(by weight) of the nanoparticle framework was the antibacterialacrylate. FIG. 1 shows the general scheme for the formation of thenanoparticle emulsion, and the amount of drug loading into thenanoparticle during the assembly process was limited by how muchsurfactant could be used, given that amounts exceeding 3 mole % of SDScaused unwanted cytotoxicity. The final crude nanoparticle emulsionscontained up to 20% of solid content (a mixture of nanoparticles and asmall amount of non-nanoparticle polymer), and only 0.2-0.6% of activeantibacterial agent inside of the nanoparticles. The resulting emulsionsare typically milky in consistency and somewhat sticky when exposed toair, causing films to form when dried, and forming coagulants withinsyringes, micro-porous filters, and gel columns that made it verydifficult to purify and use for in vivo testing.

Purification techniques that enable the removal of residual unreactedmonomers and non-nanoparticle oligomers within the cloudy emulsion wereused. Other surfactant combinations were used to try to enhance theamount of antibiotic that could be entrapped, or to alter nanoparticlesizes, without increasing overall cytotoxicity or instability of theemulsion.

FIG. 2 depicts the polyacrylate polymer that was formed that allows forthe incorporation of the bioactive drug either through covalentlybinding to the polymer backbone or encapsulating within the hydrophobicenvironment of the micelle. This in turn limits the amount of bioactivedrug that can be contained in the particle.

FIG. 3 shows that removing all acrylates except for the acrylolatedbioactive drug (or other compound) for the emulsion polymerization wouldallow for an increase in the ability to load the desired drugs/compoundswithin the micelles, and thus the final concentration in thenanoparticle emulsion. If the same limit of 15-20% of organic materialentrapped by the surfactant inside the micelles is maintained, then thefinal concentration of the drug incorporated into the nanoparticle wouldbe considerably more than the typical 0.2%-0.6% achieved using the butylacrylate/styrene polyacrylate nanoparticles. The use of onlyN-acryloylciprofloxacin as the sole monomer then would afford anadvanced polyacrylate nanoparticle emulsion, which allows for thedelivery of higher drug content. This would in return require muchsmaller volumes of the emulsion to be synthesized and used for drugdelivery.

The avoidance of using other monomers for the nanoparticle formationadditionally removes the issue of unwanted coagulation and filmformation previously observed for the poly(butyl acrylate/styrene)nanoparticle emulsions. The residual styrene and butyl acrylate andnon-particle polymers that are not encapsulated within the surfactantcould be removed by centrifugation and dialysis, however, the resultingemulsions after purification still continued to formed rubbery filmswhen dehydrated, which clogged syringe needles and filtration membranes.The use of these particular monomers was problematic in this regard andnot using them might eliminate the need to purify the ciprofloxacinacrylate emulsions.

In this Example, a new approach to preparing antibiotic-boundpolyacrylate nanoparticle emulsions is described that completelyobviates the restriction of using butyl acrylate and styrene (or otherco-monomers) to form the nanoparticle framework, and instead, uses theantibiotic compound itself as the sole acrylate monomer for thepolymerization. This technique has never been reported and is thus animportant advance in the polymer-based nanoparticle field.

Ciprofloxacin was chosen as the antibiotic for the formation of thepolyacrylate nanoparticles. The N-acryloyl derivative of commercialciprofloxacin hydrochloride was prepared for this purpose according toN-acylation procedure.

Synthesis of N-Acryloylciprofloxacin

FIG. 4 shows the synthetic scheme for preparing N-acryloylciprofloxacin, and follows as such: To a round bottom flask was added120 ml of dichloromethane, then 3.0 g (9.0 mmol) of ciprofloxacin and1.8 ml (13.5 mmol) of triethylamine. The mixture was left stirring at 0°C. for 1 hour then acryloyl chloride (1.1 ml, 13 mmol) was addeddropwise. The ice bath was removed and the reaction was left stirringovernight. The dichloromethane was added dropwise to a flask of hexane(200 ml) to cause a precipitate to form. The solid was collected byfiltration and allowed to air dry.

Yielded 2.90 g (83.7%) as a pale yellow solid. Melting point above 250°C. ¹H NMR (400 MHz, CDCl₃) δ 8.75 (s, 1H), 8.03 (d, J=12.8 Hz, 1H), 7.36(d, J=7.1 Hz, 1H), 6.60 (dd, J=16.8, 10.5 Hz, 1H), 6.35 (dd, J=16.8, 1.7Hz, 1H), 5.76 (dd, J=10.5, 1.7 Hz, 1H), 3.86 (m, 4H), 3.52 (br. s., 1H),3.33 (m, 4H), 1.38 (d, J=6.2 Hz, 2H), 1.19 (br. s., 2H).

Formation of Poly(N-Acryloylciprofloxacin) Nanoparticle Emulsion

One of the main challenges with polymerizing the desired acryloyl analogof the bioactive drug was that most of the previous antibiotics thatwere acrylolated and loaded into the nanoparticle emulsions were solids,and thus the liquid organic monomers of styrene and butyl acrylate couldbe used to pre-dissolve the small amount of the solid acrylolatedantibiotic. This was also the case with the poly(menthyl acrylate)nanoparticle emulsions, in that the non-bioactive monomer menthylacrylate was a liquid that allowed for the dissolution of the solidN-acryloyl ciprofloxacin antibiotic in order to be incorporated intomicelles during emulsion polymerization.

Attempts to use the same procedure for emulsion polymerization of theN-acrylolated ciprofloxacin monomer failed, however. Thus it wasnecessary to pre-dissolve the N-acryloyl ciprofloxacin into an organicsolvent that could easily be evaporated off during the polymerizationprocess or after the formation of the emulsions.

It was considered important to use a solvent of very low cytotoxicity toaid in the dissolution of the bioactive compounds, in case it would alsoload into the micelles along with the bioactive compound. Afterexperimentation with various common organic solvents, includingmethanol, ethanol, propylene glycol, glycerol, and ethyl acetate;dichloromethane was chosen.

Attempted Preparation of Homo Poly(N—N-Acryloylciprofloxacin)Nanoparticle Emulsions Using Water-Soluble Organic Solvents

Two liquid organic solvents were first used to aid the dissolution ofN-acryloylciprofloxacin. Propylene glycol and glycerin have very lowcytotoxicity and due to their hydrophobic nature would likely load intothe surfactant-formed micelles, and thus potentially carry in with itthe N-acryloylciprofloxacin. Though this technique would result in aco-solvent also being incorporated into the micelles, it would stillpossibly allow for formation of the poly(N-acryloylciprofloxacin)emulsion.

However, the resulting emulsions formed from the use of these solventswere not homogeneous. Due to glycerin's high viscosity, it was verydifficult to distribute and stir properly in the aqueous media. This ledto a bilayer, preventing homogeneous mixing of the resulting emulsion.The mixture was heated up to 90° C. in order to reduce the viscosity andallow for more uniform stirring and mixing with water. However, theresulting emulsions remained non-homogeneous.

Propylene glycol provided a much better carrier solvent due to its lowerviscosity. It was able to form a more uniform emulsion and would requireno modification in procedure compared to the typical one used to makepolyacrylate nanoparticle emulsions. However, the resulting emulsionswere unstable and formed a bilayer within minutes of being removed fromthe polymerization conditions. The DLS data did confirm multiplepopulations of particles within the emulsion and very low zeta potentialvalues (−5 mV to −10 mV), which confirmed the inherent instability ofthe emulsions. So these attempts did not prove effective.

Preparation of Homo Poly(N-Acryloylciprofloxacin) Nanoparticle EmulsionsUsing a Water-insoluble Solvent.

The other method investigated for polymerization of the solidN-acryloylciprofloxacin to be evenly distributed within the aqueousmixture was to pre-dissolve the compound in an organic solvent, and thenremove the organic solvent via evaporation during the emulsion processor after the emulsion formation. It was critical to completely removethe organic solvent, because most organic solvents produce cytotoxicity.

This method was attempted using methanol, ethanol, ethyl acetate, anddichloromethane. The main problem was the poor solubility of theN-acryloylciprofloxacin in most organic solvents, except fordichloromethane. Up to 500 mg/ml of N-acryloylciprofloxacin could bedissolved into dichloromethane. However, there was a critical issue thatresulted with the polymerization procedure. Typically, organics werestirred at 75° C., then the surfactant and water were added. This wouldcause the dichloromethane to rapidly evaporate. Thus the startingtemperature was adjusted to 25° C., and water and surfactant were addedto the stirring dichloromethane solution, however this resulted in anuneven distribution and clumping of the surfactant. The result was avery sticky material that separated from the water layer.

To solve this, the surfactant and water were added first at 75° C. sothat the surfactant may form micelles initially, and the dichloromethanesolution was added dropwise. However, this resulted in the near instantevaporation of the dichloromethane solvent, leaving clumps of solidN-acryloylciprofloxacin unincorporated into the micelles. The finaladjustment of the procedure is discussed in the following section,resulting in successful formation of the emulsion.

Preparation of Poly(N-Acryloylciprofloxacin) Nanoparticle Emulsions

As seen in FIG. 5, the polyacrylate emulsions were prepared using amodified protocol of the usual nanoparticle emulsion technique used inour lab. The method to form the poly(N-acryloylciprofloxacin) emulsionrequired the following procedure: to a round bottom flask was added 4 mlof deionized water, which was then stirred using a 1.25 cm (300 mg)Teflon-coated magnetic stir bar at 1000 rpm on a Corning PC-420Dmagnetic stirrer at 30° C. using a self-regulated oil bath. To this wasadded 30 mg of SDS. N-Acryloylciprofloxacin (500 mg) was dissolved in 1ml of warm dichloromethane, and this solution was added dropwise to thedeionized water-SDS mixture. A vent was placed on top of the flask byinserting a small stainless steel syringe needle through a rubber septumon the flask, under dry nitrogen, and the temperature of the mixture wasincreased at a rate of 5° C. per 30 min until reaching 90° C. Themixture was left stirring overnight at this temperature, under anatmosphere of dry nitrogen. Potassium persulfate (10 mg) was added withan additional 0.5 ml of deionized water to the stirring mixture, andleft stirring for 24 hours. The stirred emulsion was then removed fromthe oil bath and decanted into a storage vial for analysis.

FIG. 6 shows an example of a successful emulsion (on the left), forminga uniform single layer emulsion, while previous attempted emulsions (thetwo on the right) show the results of an unsuccessful emulsionpolymerization.

Dynamic Light Scattering (DLS) Analysis

Dynamic light scattering measurements were performed to test if anynanoparticles were being formed in the emulsion polymerization process.The average size and surface charge of the emulsion was analyzed on aMalvern Zetasizer nano-ZS instrument. To prepare the samples for theanalyses, the freshly-made emulsion was subjected to centrifugation at10,000 rpm for 5 min using an Eppendorf Centrifuge 5424. An aliquot ofthe liquid emulsion was then drawn and deposited into a Malverndisposable folded capillary cell DTS-1070. Each sample was analyzed intriplicate, and each data collection consisted of 1 run of 20 scans (forsize analysis) and 3 runs of 100 scans (for zeta potentialdetermination). The size distribution shows a single narrow peakindicating the uniformity of the emulsion with a single populationcentered on average at approximately 970 nm. Similarly, surface chargemeasurements indicated a highly stable emulsion, with an average of −63(±5.6) mV.

Dynamic Light Scattering (DLS) Analysis Results

As FIG. 7 demonstrates, the dynamic light scattering experimentsconfirmed the presence of a major population of nanoparticles in theemulsion, measuring on average approximately 970 nm in diameter. Ageneral trend of increasing size was observed as the amount ofN-acryloylciprofloxacin is increased in forming the polymer emulsions.In addition, the zeta potential measurements showed that the particlescarry a high surface charge of −63 (±5.6) mV. This indicates thelong-term stability of the emulsion. It is notable that thesepoly(N-acryloylciprofloxacin) nanoparticles are much larger than thosepreviously constructed with butyl acrylate-styrene co-monomers, whichroutinely measured 45-50 nm in diameter. The basis for this 20-foldincrease in size is not apparent at this time but deserves furtherinvestigation.

In Vitro Antibacterial Testing

To investigate whether the nanoparticles possess antibioticcapabilities, each crude emulsion was tested against Staphylococcusaureus (ATCC 25923) and Escherichia coli (K12) using a 96-well platebroth assay to determine the minimum inhibitory concentration (MIC).Each assay was done in triplicate.

The original stock emulsion was diluted using the Trypticase Soy Brothsolution to an initial concentration of 1.28 mg/ml of theN-acryloylciprofloxacin, then serial diluted with TSB to half theconcentration each time. A volume of 10 μl of each emulsion dilution wasadded to a well in series, resulting in a final concentration run of 64μg/ml to 0.012 μg/ml. The MIC was done in triplicates for eachbacterium, with ciprofloxacin hydrochloride being used as a positivecontrol and a blank of broth medium was used as a negative control.

Bacteria were grown overnight at 37° C. on an agar plate composed of BBLTSA II Trypticase Soy Agar (TSA) and BBL Trypticase Soy Broth (TSB) in a1:2 ratio at 4.4% concentration. A broth solution of 2.4% TSB wasinoculated using the bacteria from the agar plates, and incubated at 37°C. to reach a 0.5 McFarland standard. The bacteria were then furtherdiluted by a factor of 1000 using a broth solution of 2.4% TSB, and 190μl of the diluted bacterial solution was transferred by micropipetteinto each well. The inoculated plates were incubated at 37° C. for 16-20hours and the resulting plates were observed for growth and MIC valuesrecorded. The MIC was the lowest concentration of the antibiotic thatcompletely inhibited bacterial growth (visually) within that series ofdilutions.

Antibacterial Data for Poly (N-Acryloylciprofloxacin) Emulsions

TABLE 1 MIC values of ciprofloxacin and ciprofloxacin emulsion vs S.aureus and E. coli. S. aureus E. coli Sample (ATCC 25923) (K12) ControlCiprofloxacin 0.5 μg/ml 0.012 μg/ml Poly (N-acryloyl-ciprofloxacin) 0.5μg/ml 0.012 μg/ml emulsion

The in vitro antibacterial studies showed that the nanoparticle emulsionwas bioactive, with an MIC of 0.5 μg/ml for S. aureus and 0.012 μg/mlagainst E. coli, identical to those of ciprofloxacin itself (Table 1).The finding that these nanoparticles show antibacterial capabilitiesagainst both the gram-positive S. aureus and the gram-negative E. coliwas surprising, given that particles of such large dimensions would notbe expected to be antibacterially active.

Ciprofloxacin must enter the bacterial cell to arrive at its target,bacterial DNA gyrase. Attachment of the molecule to the polymer backboneof the nanoparticle requires it be released through hydrolysis of theamide. This occurs either outside of the cell or within the bacteriumitself if the nanoparticle can enter through the membrane. Most likelythis requires enzymatic release, as the amide functionality is adifficult one to cleave otherwise.

In Vitro Cytotoxicity of the Nanoparticle Emulsions

In vitro cell cytotoxicity was tested on two human cell lines, humancolorectal carcinoma cells HCT-116, and human embryonic kidney cells HEK293. HCT-116 cells were grown in Dulbeco's Minimum Essential Medium(DMEM) with 10% fetal bovine serum and 0.1% penicillin/streptomycin as agrowth medium for several days at 37° C. under an atmosphere of 5% CO₂to reach confluence. HEK 293 cells were grown in Eagle Minimum EssentialMedium (EMEM) with 10% fetal bovine serum and 0.1%penicillin/streptomycin as a growth medium for several days at 37° C.under an atmosphere of 5% CO₂ to reach confluence. Each cell type wasthen plated onto 96-well plates, at 50,000 cells per well at a volume of150 μl with the respective growth medium. The cells were counted using ahemocytometer and then incubated for 24 hours at 37° C. under anatmosphere of 5% CO₂.

The test emulsion was diluted using the growth medium for each celltype, and added into the wells of each test plate to give a finalconcentration of N-acryloyl ciprofloxacin of 2 mg/ml, 1 mg/ml, 0.5mg/ml, 0.25 mg/ml, 0.125 mg/ml, and 0.0625 mg/ml within a series. Thetesting was done in triplicate and one well in each triplicate was leftuntreated as the negative control for 100% growth. The plates werefurther incubated and monitored for 48 hours at 37° C. under anatmosphere of 5% CO₂. A 5 mg/ml solution of3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) insterile phosphate-buffered saline (PBS) was added to give a 10% finalconcentration in each well. The plates were then further incubated for 4hours at 37° C. under an atmosphere of 5% CO₂ to allow for the formationof the purple crystals of1-(4,5-dimethylthiazol)2-yl)-3,5-diphenylformazan. The liquid was thenaspirated from each well and 100 μl of dimethylsulfoxide (DMSO) wasadded to each well, and gently shaken for 1 minute to allow for completedissolution of the crystals. The IC₅₀ value for the assay was determinedusing a BioTek Synergy H1 hybrid plate reader at both 595 nm and 630 nm.The IC₅₀ was determined as the well with at least 50% cell viabilitycompared to the untreated control cell with 100% cell growth.

Cytotoxicity Results for Poly(N-Acryloylciprofloxacin) NanoparticleEmulsions

The observed IC₅₀ was 500 μg/ml for both the HCT-116 and HEK-293 celllines, a 1000-fold difference over the bacterial MIC value for S. aureusand greater than 40,000 for E. coli.

Imaging Nanoparticle Emulsions Using a Scanning Electron Microscope

A sample of poly(N-acryloyl ciprofloxacin) nanoparticle emulsion wasprepared for imaging using scanning electron microscope. The sampleswere initially prepared by lyophilization of the emulsion which resultedin a dry powder that could be added to the sample holder for thescanning electron microscope instrument. The samples were placed onto analuminum-coated sample holding tape, mounted onto a copper tape, placedonto the scanning electron microscope sample holder. The sample wasdiluted 1000× with deionized water, and a drop of the diluted emulsionwas placed on the conductive aluminum-coated sample holding tape. Thesample was placed in the −80° C. freezer for a few hours, thenimmediately lyophilized to dry the sample right onto the sample holdingtape to produce a more even distribution of the material.

In addition, the sample-containing tape was also sputter-coated withgold-palladium in order to increase the conductivity of the resultingsample, thus preventing or reducing the accumulation of electrons on thesurface of the sample, and resulting in distortions.

As observed in FIGS. 8 and 9, the images from the scanning electronmicroscope do not provide clear images of the spheres within theemulsion as were previously observed with butyl acrylate/styrene andpoly(menthyl acrylate) emulsions. This was thought to be the result ofthe material continuing to building up charge on the surface, thusgiving a distorted image. Attempts to overcome this effect by ensuring asmooth and conductive surface for the sample holding tape, andsputter-coating with conductive gold-palladium coating, did not improveresults. In addition, during the lyophilization process the spheres weredehydrated and deformed, thus resulting in the spheres binding to eachother and not remaining separate. This led to the increase of theoverall size when viewed from top down with the scanning electronmicroscope.

Poly(N-acryloylciprofloxacin) nanoparticle emulsions were successfullyprepared by modification of the previously reported emulsionpolymerization methodology. The main difference with this new method wasthe need to dissolve the water-insoluble antibacterial agent in anorganic solvent to permit more uniform addition into the aqueoussolution to form homogeneous emulsions. Dichloromethane provided thebest combination of solubilizing the ciprofloxacin monomer and beingvolatile enough to evaporate from the media during emulsionpolymerization at 90° C.

The increased temperature of 90° C. rather than 75° C., an increasedstir speed, and the addition of sodium dodecyl sulfate before theorganic monomers were added, provided more optimal results.Additionally, it was advantageous to let the reactions run for 48 hoursrather than the 6 hours required for the butyl acrylate-styreneco-monomer systems.

These new procedures are required mainly due to the physical propertiesof the compounds involved, and are pushing the limits and capabilitiesof the existing available equipment. Higher loading of the drug couldperhaps be possible if the mixture could be heated in a pressurizedsystem that would allow for a higher temperature to be achieved withoutboiling off the water. In addition, a mechanical stirrer able to achievea higher spin rate that the existing magnetic stir bar method would mostlikely allow for additional loading of the monomer, since it wouldprovide more uniform distribution of large quantities of the solidmonomer.

Lyophilization of the nanoparticle emulsion produced an amorphous powderthat could not be reformulated to its original emulsified state throughaddition of water. Moreover, the resulting powder remained insoluble inorganic solvents including methanol, ethanol, dichloromethane, hexane,acetone, ethyl acetate, and dimethylformamide. Extraction of the solidmaterial with methanol, ethanol, dichloromethane, hexane, acetone, orethyl acetate failed to show any trace of unreactedN-acryloylciprofloxacin upon evaporation and analysis by proton NMRspectroscopy. This confirms that the polymerization is complete, andthus all of the N-acryloylciprofloxacin is incorporated into theframework of the nanoparticle. Attempts to perform the emulsionpolymerization procedure on the free ciprofloxacin instead of theN-acryloyl derivative led to a bilayer mixture, not an emulsion, withthe layers separating within seconds after stirring was stopped.Additionally, the same procedure was attempted using N-acetylciprofloxacin as an analog similar in structure but without therequisite olefin. Once again, only an unstable mixture was formed, whichseparated into layers with a few seconds after stirring was stopped.Therefore, the acryloyl group is a prerequisite for emulsification andsubsequent nanoparticle formation.

This Example provides an aqueous nanoparticle polymer emulsion beingformed from a monomer that is the antibiotic agent itself. The emulsionis formed via a one pot reaction in water and the final antibioticpolymer is suspended in water. The emulsified nano-cipro particles areantimicrobially-active towards gram positive S. aureus and gram negativeE. coli.

The methods and nanoparticle emulsions described herein can be used toproduce other emulsions containing other antibiotics includingwater-insoluble antibiotics for delivery and effective treatment ofdrug-resistant bacterial infections.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

REFERENCES

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We claim:
 1. A homopolymer consisting of a polymerized acrylolated drugmonomer, wherein the acrylolated drug monomer is an antibiotic selectedfrom a β-lactam or a fluoroquinolone.
 2. A nanoparticle comprising thehomopolymer of claim
 1. 3. The homopolymer of claim 1, wherein theβ-lactam is benzylpenicillin, benzathine benzylpenicillin, procainebenzylpenicillin, phenoxymethylpenicillin (V), propicillin,pheneticillin, azidocillin, clometocillin, penamecillin, cloxacillin,dicloxacillin, flucloxacillin, oxacillin, nafcillin, methicillin,amoxicillin, ampicillin, pivampicillin, hetacillin, bacampicillin,metampicillin, talampicillin, epicillin, ticarcillin, carbenicillin,carindacillin, temocillin, piperacillin, azlocillin, mezlocillin,mecillinam, pivmecillinam, sulbenicillin, faropenem, ritipenem,ertapenem, antipseudomonal, doripenem, imipenem, meropenem, biapenem,panipenem, cefazolin, cefalexin, cefadroxil, cefapirin, cefazedone,cefazaflur, cefradine, cefroxadine, ceftezole, cefaloglycin,cefacetrile, cefalonium, cfaloridine, cefalotin, cefatrizine, cefaclor,cefotetan, cephamycin, cefoxitin, cefprozil, cefuroxime, cefuroximeaxetil, cefamandole, cefminox, cefonicid, ceforanide, cefotiam,cefbuperazone, cefuzonam, cefmetazole, carbacephem, loracarbef,cefixime, ceftriaxone, antipseudomonal, ceftazidime, cefoperazone,cefdinir, cefcapene, cefdaloxime, ceftizoxime, cefmenoxime, cefotaxime,cefpiramide, cefpodoxime, ceftibuten, cefditoren, cefetamet, cefodizime,cefpimizole, cefsulodin, cefteram, ceftiolene, oxacephem, flomoxef,latamoxef, cefepime, cefozopran, cefpirome, cefquinome, ceftarolinefosamil, ceftolozane, ceftobiprole, ceftiofur, cefquinome, cefovecin,aztreonam, tigemonam, carumonam, or nocardicin A.
 4. The homopolymer ofclaim 1, wherein the fluoroquinolone is enoxacin, ciprofloxacin,norfloxacin, ofloxacin, levofloxacin, trovafloxacin, gatifloxacin, ormoxifloxacin.
 5. The nanoparticle of claim 2, having a size of between600 nm to 1000 nm.
 6. The nanoparticle of claim 2, further comprising adetergent.
 7. The nanoparticle of claim 6, wherein the detergent issodium dodecyl sulfate, cetyltrimethylammonium bromide,3-(N,N-dimethylmyristylammonio)propanosulfonate, dedecanoic acid2-(2-hydroxyethoxy)ethyl ester, sodium 11-(acrylolyloxyundecan-1-yl)sulfate,N-(11-Acryloyloxyundecyl)-N-(2-hydroxyethyl)-N,N-dimethylammoniuimbromide, N-(11-Acryloyloxyundecyl)-N,N-dimethyl-N-ethylammonium bromide,3-[N,N-Diethyl-N-(3-sulfopropyl)ammonio]acrylate,2(2-Acryloyloxyethoxy)ethyl dodecanoate, or any mixture thereof.